Epigenetic alterations differ in phenotypically distinct human neuroblastoma cell lines

<p>Abstract</p> <p>Background</p> <p>Epigenetic aberrations and a CpG island methylator phenotype have been shown to be associated with poor outcomes in children with neuroblastoma (NB). Seven cancer related genes (<it>THBS-1, CASP8, HIN-1, TIG-1, BLU, SPARC</it>, and <it>HIC-1</it>) that have been shown to have epigenetic changes in adult cancers and play important roles in the regulation of angiogenesis, tumor growth, and apoptosis were analyzed to investigate the role epigenetic alterations play in determining NB phenotype.</p> <p>Methods</p> <p>Two NB cell lines (tumorigenic LA1-55n and non-tumorigenic LA1-5s) that differ in their ability to form colonies in soft agar and tumors in nude mice were used. Quantitative RNA expression analyses were performed on seven genes in LA1-5s, LA1-55n and 5-Aza-dC treated LA1-55n NB cell lines. The methylation status around <it>THBS-1, HIN-1, TIG-1 </it>and <it>CASP8 </it>promoters was examined using methylation specific PCR. Chromatin immunoprecipitation assay was used to examine histone modifications along the <it>THBS-1 </it>promoter. Luciferase assay was used to determine <it>THBS-1 </it>promoter activity. Cell proliferation assay was used to examine the effect of 5-Aza-dC on NB cell growth. The soft agar assay was used to determine the tumorigenicity.</p> <p>Results</p> <p>Promoter methylation values for <it>THBS-1</it>, <it>HIN-1</it>, <it>TIG-1</it>, and <it>CASP8 </it>were higher in LA1-55n cells compared to LA1-5s cells. Consistent with the promoter methylation status, lower levels of gene expression were detected in the LA1-55n cells. Histone marks associated with repressive chromatin states (H3K9Me3, H3K27Me3, and H3K4Me3) were identified in the <it>THBS-1 </it>promoter region in the LA1-55n cells, but not the LA1-5s cells. In contrast, the three histone codes associated with an active chromatin state (acetyl H3, acetyl H4, and H3K4Me3) were present in the <it>THBS-1 </it>promoter region in LA1-5s cells, but not the LA1-55n cells, suggesting that an accessible chromatin structure is important for <it>THBS-1 </it>expression. We also show that 5-Aza-dC treatment of LA1-55n cells alters the DNA methylation status and the histone code in the <it>THBS-1 </it>promoter modifies cell morphology, and inhibits their ability to form colonies in soft agar.</p> <p>Conclusion</p> <p>Our results suggest that epigenetic aberrations contribute to NB phenotype, and that tumorigenic properties can be inhibited by reversing the epigenetic changes with 5-Aza-dC.</p

The Prognostic Role of RASSF1A Promoter Methylation in Breast Cancer: A Meta-Analysis of Published Data

promoter methylation status and both disease free survival (DFS) and overall survival (OS) in female breast cancer.Eligible studies were identified through searching the PubMed, Web of Science and Embase databases. Studies were pooled and summary hazard ratios (HR) with corresponding confidence intervals (CIs) were calculated. Funnel plots were also carried out to evaluate publication bias. promoter methylation status with OS in 1439 patients. The hazard estimates ranged from 1.21–6.90 with a combined random-effects estimates of 3.47 (95%CI 1.44–8.34). OS reported in multivariate analysis was evaluated in four series comprising 1346 cases and the summarized random-effects HR estimate was 3.35 (95%CI 1.14–9.85). Additionally, no publication bias was detected for both OS and DFS. promoter methylation

HD-PTP Is a Catalytically Inactive Tyrosine Phosphatase Due to a Conserved Divergence in Its Phosphatase Domain

The HD-PTP protein has been described as a tumor suppressor candidate and based on its amino acid sequence, categorized as a classical non-transmembrane protein tyrosine phosphatase (PTP). To date, no HD-PTP phosphorylated substrate has been identified and controversial results concerning its catalytic activity have been recently reported

RASSF1A hypermethylation in pretreatment serum DNA of neuroblastoma patients: a prognostic marker

The tumour suppressor gene RASSF1A is known to be frequently silenced by promoter hypermethylation in neuroblastoma tumours. Here we explored the possible prognostic significance of aberrant promoter hypermethylation of RASSF1A in serum DNA samples of patients with neuroblastoma as a surrogate marker for circulating tumour cells. We analysed the methylation status of the RASSF1A gene in matched tumour and pretreatment serum DNA obtained from 68 neuroblastoma patients. Hypermethylation of RASSF1A in tumour samples was found in 64 patients (94%). In contrast, serum methylation of RASSF1A was observed in 17 patients (25%). Serum methylation of RASSF1A was found to be statistically associated with age ⩾12 months at diagnosis (P=0.002), stage 4 (P<0.001) and MYCN amplification (P<0.001). The influence of serum RASSF1A methylation on prognosis was found to be comparable with that of the currently most reliable marker, MYCN amplification on univariate analysis (hazard ratio, 9.2; 95% confidence interval (CI), 2.8–30.1; P<0.001). In multivariate analysis of survival, methylation of RASSF1A in serum had a hazard ratio of 2.4 (95% CI, 0.6–9.2), although this association did not reach statistical significance (P=0.194). These findings show that the methylation status of RASSF1A in the serum of patients with neuroblastoma has the potential to become a prognostic predictor of outcome

DLEC1 is a functional 3p22.3 tumour suppressor silenced by promoter CpG methylation in colon and gastric cancers

Promoter CpG methylation of tumour suppressor genes (TSGs) is an epigenetic biomarker for TSG identification and molecular diagnosis. We screened genome wide for novel methylated genes through methylation subtraction of a genetic demethylation model of colon cancer (double knockout of DNMT1 and DNMT3B in HCT116) and identified DLEC1 (Deleted in lung and oesophageal cancer 1), a major 3p22.3 TSG, as one of the methylated targets. We further found that DLEC1 was downregulated or silenced in most colorectal and gastric cell lines due to promoter methylation, whereas broadly expressed in normal tissues including colon and stomach, and unmethylated in expressing cell lines and immortalised normal colon epithelial cells. DLEC1 expression was reactivated through pharmacologic or genetic demethylation, indicating a DNMT1/DNMT3B-mediated methylation silencing. Aberrant methylation was further detected in primary colorectal (10 out of 34, 29%) and gastric tumours (30 out of 89, 34%), but seldom in paired normal colon (0 out of 17) and gastric (1 out of 20, 5%) samples. No correlation between DLEC1 methylation and clinical parameters of gastric cancers was found. Ectopic expression of DLEC1 in silenced HCT116 and MKN45 cells strongly inhibited their clonogenicity. Thus, DLEC1 is a functional tumour suppressor, being frequently silenced by epigenetic mechanism in gastrointestinal tumours

Long-range epigenetic silencing at 2q14.2 affects most human colorectal cancers and may have application as a non-invasive biomarker of disease

Large chromosomal regions can be suppressed in cancer cells as denoted by hypermethylation of neighbouring CpG islands and downregulation of most genes within the region. We have analysed the extent and prevalence of long-range epigenetic silencing at 2q14.2 (the first and best characterised example of coordinated epigenetic remodelling) and investigated its possible applicability as a non-invasive diagnostic marker of human colorectal cancer using different approaches and biological samples. Hypermethylation of at least one of the CpG islands analysed (EN1, SCTR, INHBB) occurred in most carcinomas (90%), with EN1 methylated in 73 and 40% of carcinomas and adenomas, respectively. Gene suppression was a common phenomenon in all the tumours analysed and affected both methylated and unmethylated genes. Detection of methylated EN1 using bisulfite treatment and melting curve (MC) analysis from stool DNA in patients and controls resulted in a predictive capacity of, 44% sensitivity in positive patients (27% of overall sensitivity) and 97% specificity. We conclude that epigenetic suppression along 2q14.2 is common to most colorectal cancers and the presence of a methylated EN1 CpG island in stool DNA might be used as biomarker of neoplastic disease

Whole Blood DNA Aberrant Methylation in Pancreatic Adenocarcinoma Shows Association with the Course of the Disease: A Pilot Study

Pancreatic tumors are usually diagnosed at an advanced stage in the progression of the disease, thus reducing the survival chances of the patients. Non-invasive early detection would greatly enhance therapy and survival rates. Toward this aim, we investigated in a pilot study the power of methylation changes in whole blood as predictive markers for the detection of pancreatic tumors. We investigated methylation levels at selected CpG sites in the CpG rich regions at the promoter regions of p16, RARbeta, TNFRSF10C, APC, ACIN1, DAPK1, 3OST2, BCL2 and CD44 in the blood of 30 pancreatic tumor patients and in the blood of 49 matching controls. In addition, we studied LINE-1 and Alu repeats using degenerate amplification approach as a surrogate marker for genome-wide methylation. The site-specific methylation measurements at selected CpG sites were done by the SIRPH method. Our results show that in the patient’s blood, tumor suppressor genes were slightly but significantly higher methylated at several CpG sites, while repeats were slightly less methylated compared to control blood. This was found to be significantly associated with higher risk for pancreatic ductal adenocarcinoma. Additionally, high methylation levels at TNFRSCF10C were associated with positive perineural spread of tumor cells, while higher methylation levels of TNFRSF10C and ACIN1 were significantly associated with shorter survival. This pilot study shows that methylation changes in blood could provide a promising method for early detection of pancreatic tumors. However, larger studies must be carried out to explore the clinical usefulness of a whole blood methylation based test for non-invasive early detection of pancreatic tumors